Method for immobilizing self-organizing material or fine particle on substrate, and substrate manufactured by using such method

ABSTRACT

A method for immobilizing a self-organizing material or fine particles on a substrate, and a substrate whereupon the self-organizing material or the fine particles are immobilized. More specifically, the method for immobilizing the fine particles including a nucleic acid (for instance, DNA or RNA) or a metal oxide on the substrate, and the substrate whereupon the nucleic acid (for example, DNA or RNA) or the metal oxide is immobilized.

TECHNICAL FIELD

The present invention relates to a method for immobilizing aself-organizing material or fine particles on a substrate, and asubstrate whereupon the self-organizing material or the fine particlesare immobilized. Specifically, the invention relates to a method forimmobilizing fine particles including a nucleic acid (for instance, DNAor RNA) or a metal oxide on a substrate, and a substrate whereupon thenucleic acid (for example, DNA or RNA) or the metal oxide isimmobilized.

BACKGROUND ART

In the medical field, there is high expectation for diagnosis, treatmentor prevention of diseases using genes (gene diagnosis). By examiningdefects or changes in causal genes of particular diseases for example,the gene diagnosis allows for diagnosis, treatment or prevention beforeonset of disease or at early stages of disease. The gene diagnosis alsorealizes what is known as tailor made medicine, which is based onrelationships between genotype and disease as revealed by human genomeanalysis. In view of this, a further development is expected for easydetection of genes and/or easy determination of genotypes.

As a tool for gene detection and/or genotype determination, a DNA chip(DNA microarray, or more generally nucleic acid immobilizing substrate)has been available that is used to hybridize a subject sample withnucleic acids immobilized on a substrate or a support. The DNA chip isconsidered to be useful in applications including not only basic medicalscience but clinical medicine, drug creation and/or preventive medicineas well.

The nucleic acid immobilizing substrate is also important for thedevelopment of nucleic acid-based nanotechnology (for example, nanowires, and nucleic acid-based nano electronics such as bio-sensors andbio-chips) (see Non-Patent Publication 1, for example).

In detection of nucleic acid based on a hybridization method, there hasbeen proposed a method in which a sample containing a nucleic acid(target DNA) complementary to a nucleic acid probe such as a DNAfragment is immobilized on a support such as a nitrocellulose film andis allowed to react with the nucleic acid probe in a solution (seeNon-Patent Publication 2, for example).

As a method by which nucleic acids used for the DNA chip are immobilizedon a substrate, a method is known that directly synthesizes a nucleicacid probe on a substrate (see Non-Patent Publication 3, for example).As another method of immobilizing nucleic acids on a support, there hasbeen known a method in which a nucleic acid probe is immobilized on asupport after it has been prepared beforehand (see Non-PatentPublication 4, for example).

In another known method, a solid-phase support that has beensurface-treated with a silane coupling agent including functional groupssuch as an amino group or an aldehyde group is covalently bonded to anucleic acid probe having modified functional groups, after spottingwith a DNA chip fabricating device (see Non-Patent Publication 5).

Specifically, the following methods have been known as the method ofbinding a self-organizing material (for example, nucleic acid) on asolid surface: A method in which a substrate surface is treated withsilane to introduce therein a vinyl group that can bind to the nucleicacid molecule (see Non-Patent Publication 6, for example); a method inwhich nucleic acids are bound to a substrate using counter ions (seeNon-Patent Publications 7-9, for example); a method in which pH valuesare chemically controlled to adjust the extent of immobilization onvarious types of substrate surfaces (see Non-Patent Publication 10, forexample); and a method in which Al₂O₃ surface is treated with Na₃PO₄solution to render the surface hydrophilic (see Non-Patent Publication11, for example). As a technique for removing organic impurities fromthe substrate surface, there has been known a method in which themolecules on the substrate surface are modified by an oxygen plasmaprocess which requires expensive equipment (see Patent Publications 1and 2, for example).

In the field of electronics, there has been report that nucleic acidsshow strong bonding, similar to covalent bond, with respect to aluminumelectrodes (see Non-Patent Publication 12, for example).

[Patent Publication 1]

WO97/38801 (published on Oct. 23, 1997)

[Patent Publication 2]

Japanese Laid-Open Patent Publication No. 2002-218976 (published on Aug.6, 2002)

[Non-Patent Publication 1]

Storhoff, J. J. and Mirkin, C. A.: Chem. Rev. 99, 1849-1862 (1999)

[Non-Patent Publication 2]

Molecular Cloning 2^(nd). Ed. (Cold Spring Harbor Press)

[Non-Patent Publication 3]

Forder, S. P. A. et al, Science, 251, 767-773 (1991)

[Non-Patent Publication 4]

Schena, M. et al., Science, 270, 467-470 (1995)

[Non-Patent Publication 5]

Geo, Z. et al., Nucleic Acid Research, 22, 5456-5465 (1994)

[Non-Patent Publication 6]

Bensimon, D. et al., Physical Review Letters 74, 23, 4754-4757 (1995)

[Non-Patent Publication 7]

Ye, J. Y. et al., Analytical Biochemistry 281, 21-25 (2000)

[Non-Patent Publication 8]

Dunlap, D. D. et al., Nucl. Acid Res. 25, 3095 (1997)

[Non-Patent Publication 9]

Lyubchenko, Y. L. et al., Proc. Natl. Acad. Sci. USA 94, 496 (1997)

[Non-Patent Publication 10]

Allemand, J. F. et al., Biophysical Journal, 73, 2064-2070 (1997)

[Non-Patent Publication 11]

Yoshida, K. et al., Biophysical Journal, 74, 1654-1657 (1998)

[Non-Patent Publication 12]

Washizu, M. et al., IEEE Trans. Industr. Appl., 31, 3, 447-456 (1995)

As an application of the method described in Non-Patent Publication 3,there has been known a method in which a nucleic acid probe issynthesized on a glass slide or a silicon substrate using aphotolithography technique, employed in fabrication of semiconductors,in combination with a solid-phase synthesis technique. A drawback of theon-chip synthesis method, however, is that it requires special equipmentand reagents for synthesizing a nucleic acid probe on the substrate, andthat it can synthesize nucleic acid probes of only about several tenbases long.

As an application of the method described in Non-Patent Publication 4, amethod is known in which a nucleic acid probe that has been prepared inadvance using a DNA chip fabricating device is spotted for electrostaticbonding on a surface of a solid-phase support that has beensurface-treated with poly-L-lysine or the like. While such anelectrostatic bonding method of nucleic acid probe allows forimmobilization of long nucleic acid probes, it lacks reproducibility dueto procedures of hybridization reactions.

The method described in Non-Patent Publication 5 can be used to bond thenucleic acid probe onto a solid-phase support relatively firmly.However, preparation of nucleic acid probe requires the tediousprocedures of PCR amplification using oligonucleotides with modifiedfunctional groups. Further, due to difficulties of the method inpreparing a nucleic acid probe extending several thousand bases,identification of long nucleic acid probes is inevitably difficult.

As described above, as the technique for binding nucleic acid moleculeson a substrate surface, there have been used (1) a method of modifyingmolecules on a substrate surface, or (2) a method of subjecting asubstrate surface with a plasma process. However, the method employingmolecule modification requires many steps and large equipment. Likewise,the plasma process calls for large-scale equipment and large cost. Thatis, it has not been possible with the conventional techniques todirectly bind the nucleic acid molecules, both easily and inexpensively,on a substrate surface.

In studying DNA structures and a complex thereof, or electricalcharacteristics of DNA structure, the inventors of the present inventionfound that it was important to immobilize or extend DNA on an atomicallyflat substrate in order to rule out the influence of substratestructure. Immobilization of DNA is well researched. Mica, glass, gold,and highly oriented pyrolytic graphite (HOPG) are commonly used as thesubstrate. Mica and HOPG can be cut very easily to provide an atomicallyflat substrate. However, cations (for example, magnesium ion or nickelion) are required in order to hold molecules on the substrate made ofthese materials. Glass substrates also require surface modification. Forexample, DNA is immobilized using aminopropyl triethoxysilane (APS). Bycoating the glass surface, APS forms a positively charged layer withamino groups. The DNA with the phosphate groups (negatively charged)around the double helix is therefore adsorbed on the substrate by theelectrostatic force. Gold particles can be used for DNA with thiol ends,because gold can form a covalent bond with the thiol group.

The present invention was made in view of the foregoing problems, and anobject of the present invention is to provide a self-organizing materialimmobilizing substrate that can immobilize a self-organizing material ona substrate surface in a controlled manner from low density to highdensity, and that can be manufactured at low cost. Specifically, it isan object of the present invention to provide a method for convenientlyimmobilizing DNA and a method for extending DNA without chemical surfacemodification (for example, APS process) using other molecules, and asubstrate fabricated by such methods. It is another object of thepresent invention to provide a method for one-dimensionally arrangingfine particles on a substrate surface in a desired shape within a squaremicrometer.

DISCLOSURE OF INVENTION

A method for immobilizing a self-organizing material on a substrate ofmetal oxide according to the present invention includes the steps of:

applying to the substrate an acid solution capable of introducing ahydroxy group on a surface of the substrate; and

applying a solution containing the self-organizing material onto thesubstrate after the acid solution is removed from the substrate.

In a method for immobilizing a self-organizing material on a substrateof metal oxide according to the present invention, it is preferable thatthe self-organizing material be a nucleic acid, a protein, an aminoacid, a lipid, or a sugar.

In a method for immobilizing a self-organizing material on a substrateof metal oxide according to the present invention, it is preferable thatthe metal oxide be Al₂O₃, ZnO, TiO₂, SiO₂, ZrO₂, SrTiO₂, LaAlO₃, Y₂O₃,MgO, GGG, YIG, LiTaO, LiNbO, KTaO₃, KNbO₃, or NdGaO₃.

It is preferable that a method for immobilizing a self-organizingmaterial on a substrate of metal oxide according to the presentinvention further include the step of drying the self-organizingmaterial-containing solution applied to the substrate.

In a method for immobilizing a self-organizing material on a substrateof metal oxide according to the present invention, it is preferable thatthe drying step be performed by blowing a dry inert gas or air.

A method for arranging fine particles on a substrate of metal oxideaccording to the present invention includes the steps of:

applying an acid solution to the substrate;

obtaining a mixed solution by mixing a solution containing the fineparticles with a solution containing a self-organizing material;

applying the mixed solution to the substrate after the acid solution isremoved from the substrate; and

drying the mixed solution applied to the substrate.

In a method for arranging fine particles on a substrate of metal oxideaccording to the present invention, it is preferable that the fineparticles have the same charge as the solution containing theself-organizing material.

In a method for arranging fine particles on a substrate of metal oxideaccording to the present invention, it is preferable that theself-organizing material be a nucleic acid, a protein, an amino acid, alipid, or a sugar.

In a method for arranging fine particles on a substrate of metal oxideaccording to the present invention, it is preferable that the metaloxide be Al₂O₃, ZnO, TiO₂, SiO₂, ZrO₂, SrTiO₂, LaAlO₃, Y₂O₃, MgO, GGG,YIG, LiTaO, LiNbO, KTaO₃, KNbO₃, or NdGaO₃.

In a method for arranging fine particles on a substrate of metal oxideaccording to the present invention, it is preferable that the fineparticles comprise gold, silver, platinum, palladium, iridium, rhodium,osmium, ruthenium, nickel, cobalt, indium, copper, TiO₂, or BaTiO₃.

In a method for arranging fine particles on a substrate of metal oxideaccording to the present invention, it is preferable that the fineparticles have a particle diameter in a range of 1 nm to 100 nm.

In a method for arranging fine particles on a substrate of metal oxideaccording to the present invention, it is preferable that the dryingstep be performed by blowing a dry inert gas or air.

A method for arranging fine particles on a substrate in a desired shapeaccording to the present invention includes the steps of:

immobilizing on the substrate a substance capable of binding to aself-organizing material;

imprinting the substance with a mold that has been formed with desiredirregularities, so as to pattern the substance in a desired shape;

obtaining a mixed solution by mixing a solution containing the fineparticles with a solution containing the self-organizing material;

applying the mixed solution to the patterned substance; and

drying the mixed solution applied to the substance.

In a method for arranging fine particles on a substrate in a desiredshape according to the present invention, it is preferable that the fineparticles have the same charge as the solution containing theself-organizing material.

In a method for arranging fine particles on a substrate in a desiredshape according to the present invention, it is preferable that the fineparticles comprise gold, silver, platinum, palladium, iridium, rhodium,osmium, ruthenium, nickel, cobalt, indium, copper, TiO₂, or BaTiO₃.

In a method for arranging fine particles on a substrate in a desiredshape according to the present invention, it is preferable that the fineparticles have a particle diameter in a range of 1 nm to 100 nm.

In a method for arranging fine particles on a substrate in a desiredshape according to the present invention, it is preferable that theself-organizing material be a nucleic acid, a protein, an amino acid, alipid, or a sugar.

In a method for arranging fine particles on a substrate in a desiredshape according to the present invention, it is preferable that thesubstance be poly-L-lysine or aminosilane.

In a method for arranging fine particles on a substrate in a desiredshape according to the present invention, it is preferable that thedrying step be performed by blowing a dry inert gas or air.

In a substrate on which a self-organizing material is immobilizedaccording to the present invention, the self-organizing material bindsto a hydroxy group that has been formed on a surface of the substrate byacid treatment of the substrate. It is preferable that theself-organizing material form a network structure on the substrate.

In a substrate on which a self-organizing material is immobilizedaccording to the present invention, it is preferable that theself-organizing material be a nucleic acid, a protein, an amino acid, alipid, or a sugar.

In a substrate on which a self-organizing material is immobilizedaccording to the present invention, it is preferable that the substratebe a metal oxide selected from Al₂O₃, ZnO, TiO₂, SiO₂, ZrO₂, SrTiO₂,LaAlO₃, Y₂O₃, MgO, GGG, YIG, LiTaO, LiNbO, KTaO₃, KNbO₃, or NdGaO₃.

In a substrate on which a self-organizing material is immobilizedaccording to the present invention, it is preferable that theself-organizing material carry fine particles. In this case, in asubstrate on which a self-organizing material is immobilized accordingto the present invention, it is more preferable that the self-organizingmaterial form a network structure on the substrate, and that the fineparticles be carried on the network structure of the self-organizingmaterial in portions lying on edges of a step structure on the surfaceof the substrate.

In a substrate on which a self-organizing material is immobilizedaccording to the present invention, it is preferable that the fineparticles comprise gold, silver, platinum, palladium, iridium, rhodium,osmium, ruthenium, nickel, cobalt, indium, copper, TiO₂, or BaTiO₃.

In a substrate on which a self-organizing material is immobilizedaccording to the present invention, it is preferable that the fineparticles have a particle diameter in a range of 1 nm to 100 nm.

In a substrate carrying fine particles in a desired shape according tothe present invention, a substance capable of binding to aself-organizing material is immobilized on the substrate, and whereinthe substance is patterned into a desired pattern and carries the fineparticles.

In a substrate carrying fine particles in a desired shape according tothe present invention, it is preferable that the fine particles comprisegold, silver, platinum, palladium, iridium, rhodium, osmium, ruthenium,nickel, cobalt, indium, copper, TiO₂, or BaTiO₃.

In a substrate carrying fine particles in a desired shape according tothe present invention, it is preferable that the fine particles have aparticle diameter in a range of 1 nm to 100 nm.

In a substrate carrying fine particles in a desired shape according tothe present invention, it is preferable that the self-organizingmaterial be a nucleic acid, a protein, an amino acid, a lipid, or asugar.

In a substrate carrying fine particles in a desired shape according tothe present invention, it is preferable that the substance bepoly-L-lysine or aminosilane.

A method for binding fine particles to a self-organizing materialaccording to the present invention includes the steps of:

obtaining a mixed solution by mixing a solution containing the fineparticles with a solution containing the self-organizing material;

applying the mixed solution to a substrate; and

drying the mixed solution applied to the substrate.

In a method for binding fine particles to a self-organizing materialaccording to the present invention, it is preferable that the fineparticles have the same charge as the solution containing theself-organizing material.

In a method for binding fine particles to a self-organizing materialaccording to the present invention, it is preferable that theself-organizing material be a nucleic acid, a protein, an amino acid, alipid, or a sugar.

In a method for binding fine particles to a self-organizing materialaccording to the present invention, it is preferable that the fineparticles comprise gold, silver, platinum, palladium, iridium, rhodium,osmium, ruthenium, nickel, cobalt, indium, copper, TiO₂, or BaTiO₃.

In a method for binding fine particles to a self-organizing materialaccording to the present invention, it is preferable that the fineparticles have a particle diameter in a range of 1 nm to 100 nm.

In a method for binding fine particles to a self-organizing materialaccording to the present invention, it is preferable that the dryingstep be performed by blowing a dry inert gas or air.

Additional objects, features, and strengths of the present inventionwill be made clear by the description below. Further, the advantages ofthe present invention will be evident from the following explanation inreference to the drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 a is a diagram showing an AFM image of Al₂O₃ surface that hasbeen treated with an acid solution and is flat at atomic level: Scalebar 100 nm.

FIG. 1 b is a diagram showing an AFM image of SrTiO₂ surface that hasnot been treated with an acid solution and is flat at atomic level.

FIG. 1 c is a diagram showing an AFM image of SrTiO₂ surface that hasbeen treated with an acid solution.

FIG. 2 a is a diagram showing DNA molecules that were extended under lowconcentration conditions on an acid-treated Al₂O₃ substrate: Scale bar 1μm.

FIG. 2 b is a diagram showing a network structure of DNA that was formedunder high concentration conditions on an acid-treated Al₂O₃ substrate:Scale bar 1 μm.

FIG. 2 c is a diagram showing slightly extended DNA molecules under highconcentration conditions on an Al₂O₃ substrate that was not subjected toacid treatment: Scale bar 1 μm.

FIG. 3 is a graph representing a subtractive spectrum of infraredmeasurement performed before and after acid treatment, showing that theabsorption peak at about 3500 cm⁻¹ is due to the hydroxy group on asubstrate surface.

FIG. 4 a is a diagram showing a fluorescent image of DNA moleculesimmobilized on a surface of an APS-coated glass substrate.

FIG. 4 b is a diagram showing a fluorescent image of DNA moleculesimmobilized on a surface of an acid-treated Al₂O₃ substrate.

FIG. 5 a is a diagram showing adsorption of DNA on an Al₂O₃ substrate.

FIG. 5 b is a diagram showing adsorption of gold nanoparticles on anAl₂O₃ substrate.

FIG. 6( a) is a diagram showing arrangements of gold nanoparticles thatare formed on an Al₂O₃ substrate by removing a mixed solution of DNAsolution and gold nanoparticle solution applied to the substrate, themixed solution containing DNA and gold nanoparticles at a ratio of about1:1 (moles of DNA/numbers of gold nanoparticles).

FIG. 6( b) is a diagram showing arrangements of gold nanoparticles thatare formed on an Al₂O₃ substrate by removing a mixed solution of DNAsolution and gold nanoparticle solution applied to the substrate, themixed solution containing DNA and gold nanoparticles at a ratio of about10:1 (moles of DNA/numbers of gold nanoparticles).

FIG. 6( c) is a diagram showing arrangements of gold nanoparticles thatare formed on an Al₂O₃ substrate by removing a mixed solution of DNAsolution and gold nanoparticle solution applied to the substrate, themixed solution containing DNA and gold nanoparticles at a ratio of about2:1 (moles of DNA/numbers of gold nanoparticles).

FIG. 6( d) is a diagram showing arrangements of gold nanoparticles thatare formed on an Al₂O₃ substrate by removing a mixed solution of DNAsolution and gold nanoparticle solution applied to the substrate, themixed solution containing DNA and gold nanoparticles at a ratio of about0.5:1 (moles of DNA/numbers of gold nanoparticles).

FIG. 7 a is a diagram schematically showing a step structure of theAl₂O₃ substrate, and DNA and gold nanoparticles adsorbed on the Al₂O₃substrate using a mixed solution of DNA solution and gold nanoparticlesolution.

FIG. 7 b is a diagram showing an AFM image (1 μm×1.2 μm) of goldnanoparticles arranged on the Al₂O₃ substrate using a mixed solution ofDNA solution and gold nanoparticle solution.

FIG. 8 is a diagram schematically showing processes by which goldnanoparticles are arranged on an Al₂O₃ substrate.

FIG. 9 is a diagram showing procedures of a fabrication method of aself-organizing material patterning substrate according to the presentinvention.

FIG. 10 a is a diagram showing a result of observation offluorescent-stained DNA immobilized on a specific pattern of aself-organizing material patterning substrate according to the presentinvention.

FIG. 10 b is a diagram showing a result of observation offluorescent-stained DNA immobilized on a specific pattern of aself-organizing material patterning substrate according to the presentinvention.

FIG. 10 c is a diagram showing a result of observation offluorescent-stained DNA immobilized on a specific pattern of aself-organizing material patterning substrate according to the presentinvention.

FIG. 10 d is a diagram showing a result of observation offluorescent-stained DNA immobilized on a specific pattern of aself-organizing material patterning substrate according to the presentinvention.

FIG. 11 a is a diagram showing a result of observation of a mold patternwith square grids.

FIG. 11 b is a diagram showing a result of observation of a DNA patternof a substrate immobilizing DNA on the mold of FIG. 11 a.

FIG. 11 c is a diagram showing a result of observation of a pattern inwhich rectangular molds are formed in a mold with square grids.

FIG. 11 d is a diagram showing a result of observation of a DNA patternof a substrate immobilizing DNA on the mold of FIG. 11 c.

FIG. 12 a is a diagram showing a result of atomic force microscopeobservation of a self-organizing material patterning substrate beforemodification with gold colloids.

FIG. 12 b is a diagram showing a result of atomic force microscopeobservation of a self-organizing material patterning substrate aftermodification with gold colloids.

FIG. 13 a is a diagram according to a comparative example of the presentinvention, showing gold particles adsorbed in a substrate when the DNAconcentration used to fabricate a substrate according to the presentinvention is doubled.

FIG. 13 b is a diagram according to a comparative example of the presentinvention, showing gold particles adsorbed in a substrate when the DNAconcentration used to fabricate a substrate according to the presentinvention is reduced in half.

BEST MODE FOR CARRYING OUT THE INVENTION

In studying immobilization of DNA molecules on a metal oxide substratetreated with an acid solution, the inventors of the present inventionconfirmed an increase in the hydroxy group on the acid-treated metaloxide substrate in an infrared analysis performed on the substrate.Further, by observing an increase of DNA molecule adsorbed on theacid-treated substrate using an atomic force microscope, it was foundthat the DNA structure on the substrate was affected by theconcentration of a DNA solution. Concerning the effect of acid treatmenton DNA binding ability, a comparison was made using a commerciallyavailable aminopropyl triethoxysilane (APS)-coated glass substrate usedto manufacture DNA-binding substrates. Fluorescent microscopeobservation of DNA adsorbed on the metal oxide substrate hadsubstantially the same result as that using the APS-treated glasssubstrate. It should be noted here that the commercially available glass(APS glass) with the surface coating for DNA binding has NH₃ ₊ groupsintroduced on the surface. This draws the phosphate backbone (negativecharge) of the DNA and immobilizes the DNA on the substrate surface.

The inventors of the present invention found that a self-organizingmaterial could be immobilized on a metal oxide substrate with a hydroxygroup introduced on a substrate surface by the reaction of the substratewith an acid solution.

As described above, Non-Patent Publication 11 describes a method inwhich a surface is rendered hydrophilic by the treatment of Al₂O₃surface with a Na₃PO₄ solution. The method described in Non-PatentPublication 11 is intended to improve hydrophilicity by applying asodium phosphate aqueous solution onto the substrate, and thispublication suggests that a layer of sodium phosphate is formed on asubstrate surface. That is, the substrate fabricated by the methoddescribed in Non-Patent Publication 11 has a surface structure thatgreatly differs from that of the present invention in which “a hydroxygroup is introduced on a substrate surface.”

In one embodiment, the present invention provides a method forimmobilizing a self-organizing material on a substrate including a metaloxide. In one aspect, a method according to the present inventionpreferably includes the step of applying an acid solution to asubstrate; and the step of applying a solution containing aself-organizing material to the substrate after the acid solution isremoved. In another aspect, the substrate used in the embodiment ispreferably made of metal oxide.

As used herein, a “self-organizing material” is intended a substancecapable of forming a structure by spontaneous aggregation of largenumbers of molecules (i.e., a substance with self-organizingcapability). Examples of a self-organizing material include nucleicacids such as DNA and RNA, bio-molecules such as proteins, amino acids,lipids, and sugars, as well as cells and tissue slices. Nucleic acids(DNA or RNA) are preferable. In applying the self-organizing material onthe substrate, it is preferable that the self-organizing material be inthe form of an aqueous solution. The concentration of theself-organizing material contained in a solution is not particularlylimited. A concentration range of 10 ng/μl to 10 μg/μl is preferable,and a concentration range of 150 ng/μl to 1250 ng/μl used in Examplesbelow is more preferable.

As used herein, the “acid solution” is not particularly limited as longas it is an acidic solution that does not dissolve the substrate (forexample, a mixture of hydrogen peroxide solution and hydrochloric acidsolution), and that can introduce the hydroxy group on a surface of thesubstrate used in the present invention.

As used herein, the “metal oxide” is not particularly limited as long asit is an oxide of a stable structure. Preferably, the metal oxide isselected from the group consisting of: Al₂O₃, ZnO, TiO₂, SiO₂, ZrO₂,SrTiO₂, LaAlO₃, Y₂O₃, MgO, GGG, YIG, LiTaO, LiNbO, KTaO₃, KNbO₃, andNdGaO₃. Al₂O₃ (α-Al₂O₃ (0001)) is most preferable.

Al₂O₃ has the following advantages over other substrates (for example,mica, glass, HOPG): (1) there is a highly established method ofobtaining an atomically flat surface by heat annealing in air; (2) thesurface is very stable even under atmospheric pressure; (3) it isoptically transparent for visible light; (4) it is an electricalinsulator; and (5) it is widely used in industry as is SAW element,which makes it possible to obtain a high-quality single crystals atrelatively low cost. Al₂O₃ therefore has many advantages in practicalapplications.

While the present invention has been demonstrated based on examplesusing Al₂O₃ as the substrate, a person ordinary skill in the art fromthe teaching of the present invention will readily understand that thesubstrate is not limited to Al₂O₃ to obtain the effects of the presentinvention, and that desired properties and effects can be obtained aslong as the substrate includes a metal oxide.

The inventors of the present invention also found that the amount ofself-organizing material immobilized on the metal oxide substrate couldbe increased by introducing a hydroxy group on a substrate surface bythe reaction of the substrate with an acid solution, and thatarrangement of gold fine particles bound to the self-organizing materialcould be controlled by blowing a dry inert gas or air when immobilizingthe self-organizing material.

In one embodiment, the present invention provides a method for arrangingfine particles on the substrate including a metal oxide. In one aspect,a method according to the present embodiment preferably includes thesteps of: applying an acid solution onto a substrate; obtaining a mixedsolution by mixing a solution containing fine particles with a solutioncontaining a self-organizing material; applying the mixed solution ontothe substrate after the acid solution is removed; and drying the mixedsolution applied to the substrate. In another aspect, a substrate usedin the embodiment is preferably made of metal oxide. In a methodaccording to the embodiment, the fine particles may have the chargedifferent from that of the self-organizing material or may beelectrically neutral. Preferably, the fine particles have the samecharge as the solution containing the self-organizing material.

As used in the present invention, the “fine particles” are usedinterchangeably with “nanoparticles,” and are preferably made of gold,silver, platinum, palladium, iridium, rhodium, osmium, ruthenium,nickel, cobalt, indium, copper, TiO₂, or BaTiO₃. Preferably, the fineparticles are of the size (particles diameter of 1 nm to 100 nm) thatcan form a colloidal solution.

The inventors of the present invention also found that the patterning ofself-organizing material developed by the inventors were applicable tothe present invention.

In one embodiment, the present invention provides a method of arrangingfine particles on the substrate in a desired shape. In one aspect, amethod according to the present invention includes the steps of:immobilizing on a substrate a substance that can bind to aself-organizing material; patterning the substance in a desired shape byimprinting, using a mold having desired irregularities; obtaining amixed solution by mixing a solution containing fine particles with asolution containing a self-organizing material; applying the mixedsolution on the patterned substrate; and drying the mixed solutionapplied to the substrate. In a method according to the presentembodiment, the fine particles may have the charge different from thatof the self-organizing material or may be electrically neutral.Preferably, the fine particles have the same charge as the solutioncontaining the self-organizing material.

As used herein, the “substance that can bind to a self-organizingmaterial” is not particularly limited as long as it can bind to theself-organizing material having self-organizing capability. Preferably,the substance includes poly-L-lysine or aminosilane (for example, APS).More preferably, the substance is made of poly-L-lysine or aminosilane(for example, APS). As used herein, the term “aminosilane” is intended amolecule that can bind to the OH group on a surface of the substrate(for example, glass, silicon, Al₂O₃) such that the amino group thatbinds to the self-organizing material (for example, DNA) is presented onthe outermost surface of the substrate.

Poly-L-lysine is known to bind to DNA (B. Xu., S. Wiehle., J A. Roth.,and R J. Cristiano., Gene Therapy (5), 1235-1243, 1998), and thereforecan be suitably used to immobilize the self-organizing material (DNA inparticular). The degree of polymerization of poly-L-lysine is notparticularly limited. However, since the binding of poly-L-lysine withDNA is assumed to occur based on the electrostatic interaction betweenthe negative charge due to the phosphate group of the DNA and thepositive charge due to the protonated amino acid of poly-L-lysine, it ispreferable that the amino groups constituting the binding sites in thepoly-L-lysine be interspaced with some distance. Considering this, it ispreferable that the degree of polymerization be, but is not limited to,about 20,000.

Aminosilane is widely used for immobilization of bio-substances, and iscapable of immobilizing substances with self-organizing capability suchas proteins, cells, and tissue slices, in addition to DNA. Thus, byusing aminosilane as an immobilizing layer, the substances withself-organizing capability can be immobilized on the substrate in anypattern. For example, a biotransmission circuit can be formed byartificially patterning neurons.

When immobilizing on the substrate a substance that can bind to theself-organizing material, it is preferable that the substrate be formedwith an immobilizing layer including the substance that can bind to theself-organizing material, and that the immobilizing layer be formed onthe substrate by a technique such as coating or dipping, in order toreliably immobilize the self-organizing material on the substrate.

When using a patterning technique that employs imprinting, theself-organizing material is not necessarily required to be applied inthe form of an aqueous solution, as long as the self-organizing materialcan fill the recesses of the irregular pattern of the immobilizing layerwithout being denatured.

When the immobilizing layer is a thin film, a method of forming thethin-film layer on the substrate is not particularly limited and variousconventional methods can be used. For example, a spin coating method ordipping method may be used. The material of the substrate is notparticularly limited as long as it allows for formation of a thin-filmlayer containing a material having binding ability. For example, thesubstrate may be an insulating substrate made of glass, resin, orsilicon, or may be a semiconductor substrate or a conductive substrate.

By an imprint process, the irregular pattern of the mold can betransferred onto the immobilizing layer formed on the substrate. Themold has been processed into an irregular pattern of a desired shape,and the raised portions of the mold are reflected in the final shape ofthe self-organizing material.

The material of the mold used in the present embodiment is notparticularly limited. Preferably, silicon or silicon dioxide is usedaccording to established microfabrication techniques (for example,lithography). The mold can be processed by methods known in the art. Inone preferable method for example, a resist (UV-sensitive organic film)is coated on a thermally-oxidized silicon film and the resist ispatterned by directly delineating it with an electron beam, and thisfollowed by dry etching using the resist as a mask.

For the transfer of the irregular pattern of the mold, known imprintingtechniques (for example, such as thermal cycle nanoimprint lithographyor photo nanoimprint lithography) may be used.

Temperature, pressure, time, or other conditions under which theirregular pattern of the mold is imprinted on the immobilizing layer maybe readily decided by a person ordinary skill in the art, taking intoaccount such factors as a throughput reduction due to required time forraising or lowering temperature of the immobilizing layer, a change indimensions of the immobilizing layer due to temperature changes,accuracy of transfer patterns, and alignment inaccuracy due to thermalexpansion.

After the transfer, the mold is detached from the substrate to obtainthe immobilizing layer with the irregular pattern. When thermal cyclenanoimprint lithography is used for example, the mold can be detachedfrom the substrate after the immobilizing layer has been hardened bylowering the temperature of the immobilizing layer. When using photonanoimprint lithography, the mold can be detached from the substrateafter the immobilizing layer has been hardened by irradiation of UVlight.

In one embodiment, the present invention provides a substrate having aself-organizing material immobilized thereon. In one aspect, it ispreferable in a substrate according to the present embodiment that theself-organizing material be bound to the hydroxy group that has beenformed on the substrate surface by an acid treatment of the substrate.In another aspect, it is preferable in a substrate according to thepresent embodiment that the self-organizing material carry fineparticles. It is preferable that the fine particles be made of gold,silver, platinum, palladium, iridium, rhodium, osmium, ruthenium,nickel, cobalt, indium, copper, TiO₂, or BaTiO₃. Preferably, the fineparticles are of the size (particles diameter of 1 nm to 100 nm) thatcan form a colloidal solution.

In one embodiment, the present invention provides a substrate of adesired shape carrying fine particles. In one aspect, it is preferablein a substrate according to the present embodiment that a substance thatcan bind to the self-organizing material be immobilized on thesubstrate, and that the substance be patterned into a desired shape andcarry fine particles. In another aspect, it is preferable in a substrateaccording to the present embodiment that the fine particles be made ofgold, silver, platinum, palladium, iridium, rhodium, osmium, ruthenium,nickel, cobalt, indium, copper, TiO₂, or BaTiO₃. Preferably, the fineparticles are of the size (particles diameter of 1 nm to 100 nm) thatcan form a colloidal solution.

When the self-organizing material is nucleic acid (DNA or RNA), asubstrate according to the present invention can be used as a functionalconductive material. Nucleic acids (DNA or RNA) are functionalconductive material with characteristic energy levels and specificproperties. When doped in DNA or RNA, elements of specific species showconsiderable changes in their electrical properties.

In the base portion of DNA, a pigment can be intercalated by paistacking. The base portion of RNA can interact with a pigment. Thus, inDNA with an intercalated pigment or RNA interacting with a pigment,irradiation of light excites the pigment and renders the DNA strand orRNA strand electrically conductive.

It is therefore possible to use the substrate as a functionallyconductive material by intercalating a pigment to the DNA immobilized onthe substrate, or by causing a pigment to interact with the RNAimmobilized in the patterning substrate of the self-organizing material.That is, an optical switching material can be constructed that emitslight according to the DNA or RNA pattern arranged on the substrate.

The pigment is not particularly limited; however, for reasons that DNAor RNA and the photo-excited pigment have close energy levels, use ofacridine orange is preferable. Representative examples of intercalatorinclude: ethidium bromide, octadecyl acridine orange, ferrocenylnaphthalene diimide, β-carboline, anthraquinone, a bisacridine viologenderivative, and a Ru complex.

A substrate according to the present invention can be used to prepare aphotomask. A photomask refers to a mask blank with a patterned image(see Glossary of Technical Terms in Japanese Industrial Standards, 5thedition, page 1954, Japanese Standards Association), and aninorganic/metal material as represented by a chrome mask is usedtherefor.

When using a substrate according to the present invention as aphotomask, the self-organizing material as a bio-material (for example,nucleic acid such as DNA or RNA) can be removed from the photomask atonce by a chemical treatment using acids or alkalis, or a heattreatment. Thus, with a photomask using a substrate according to thepresent invention, the DNA or RNA immobilized in any pattern on thesubstrate can be used for microfabrication. Further, by degrading andremoving the immobilized DNA or RNA after the process, the whole processcan be simplified and various improvements, such as improved yield, canbe expected.

The inventors of the present invention also found that theself-organizing material and the fine particles could directly bindtogether even when they have the same charge.

In one embodiment, the present invention provides a method for bindingfine particles to a self-organizing material. In one aspect, a methodaccording to the present embodiment preferably includes the steps of:obtaining a mixed solution by mixing a solution containing fineparticles with a solution containing a self-organizing material;applying the mixed solution onto a substrate; and drying the mixedsolution applied to the substrate. In a method according to theembodiment, the fine particles may have the charge different from thatof the self-organizing material or may be electrically neutral.Preferably, the fine particles have the same charge as the solutioncontaining the self-organizing material.

In a method for arranging fine particles on a substrate of metal oxideaccording to the present invention, the drying step may be performed bya method using a centrifugal separator, a method using a spin coater, ora method of blowing a dry inert gas or air. However, it is preferablethat the drying method be performed by a method of blowing a drynitrogen gas. As used herein, the “inert gas” is intended nitrogen gasor argon gas.

As described above, the inventors of the present invention accomplishedthe present invention by finding a method of conveniently immobilizingDNA and a method of extending DNA, using an Al₂O₃ (α-Al₂O₃ (0001))substrate and without any chemical surface modification (for example,APS process) that uses other molecules on the substrate. The novelmethod relies on hydrophilicity on a substrate surface, which isenhanced by the increased amount of hydroxy group by the acid solutionprocess. Immobilization and extension of DNA can be enhanced using thissimple method of cleaning the Al₂O₃ substrate.

The present invention is not limited to the description of theembodiments above, but may be altered by a skilled person within thescope of the claims. An embodiment based on a proper combination oftechnical means disclosed in different embodiments is encompassed in thetechnical scope of the present invention.

The following will describe the present invention in more detail basedon Examples. Various changes, revisions, and modifications are possibleby a person ordinary skill in the art within the scope of the claims setforth below.

EXAMPLES Example 1

The Al₂O₃ substrate used in this study was processed as follows. Thesubstrate was first heated for 5 minutes in a boiled phosphate solution,and then rinsed with ultrapure water. The substrate was then annealedfor 2 hours at 1200° C. to 1300° C. under atmospheric pressure, in orderto obtain an atomically flat surface. The surface of the Al₂O₃ substrateafter annealing process had an atomically flat step structure.

A 5:43 mixture of hydrogen peroxide solution and ultrapure water wasboiled and hydrochloric acid was added thereto (2/50 of the totalvolume). After bubbles had been formed in the mixture, the Al₂O₃substrate with the step structure was dipped in the mixture for 5minutes and then rinsed with ultrapure water.

FIG. 1 a is a result of observation of a surface structure of Al₂O₃substrate after acid treatment, using an atomic force microscope(Nanoscope IV, DI Instruments). It can be seen that rinsing did notalter the surface structure.

A SrTiO₂ substrate was annealed for 3 hours at 1000° C. under oxygenflow conditions. The substrate surface after annealing process had anatomically flat step structure.

A 5:43 mixture of hydrogen peroxide solution and ultrapure water wasboiled and hydrochloric acid was added thereto (2/50 of the totalvolume). After bubbles had been formed in the mixture, the SrTiO₂substrate with the step structure was dipped in the mixture for 5minutes and then rinsed with ultrapure water.

FIGS. 1 b and 1 c are results of observation of a surface structure ofSrTiO₂ substrate using an atomic force microscope (Nanoscope IV, DIInstruments). FIG. 1 b shows a SrTiO₂ surface before washing, and FIG. 1c shows a SrTiO₂ surface after washing. Before washing, the surface hada flat step structure with a height of about 0.4 nm. The washing processetched the surface and generated recesses as deep as about 10 nm.

The foregoing results show that the Al₂O₃ substrate, which does notundergo changes in the surface structure by the washing process is moresuitable.

Example 2

In order to immobilize and extend DNA, λ DNA solution (Takara Corp.) wasdropped on a substrate that had been subjected to a hydrophilictreatment. After 5 minutes, the substrate was dried by blowing a drynitrogen gas onto a substrate surface. Under controlled gas pressure ofabout 0.1 MPa, the gas was blown from about a 45-degree angle withrespect to the substrate surface. The DNA solution was dialyzedovernight prior to use. For comparison, substrates with and without acidtreatment were used. FIG. 2 is an AFM image of DNA immobilized on asubstrate surface at varying DNA concentrations. FIGS. 2 a and 2 b showresults from surfaces with acid treatment. FIG. 2 c shows a result froma surface subjected to annealing process alone. In a low-concentrationsolution (3 μg/ml), the isolated DNA molecules were extended andimmobilized individually on the surface (FIG. 2 a). Underhigh-concentration conditions (400 μg/ml), a network structure of DNAwas observed (FIG. 2 b). In contrast, on the substrate without acidtreatment, slightly extended DNA molecules were observed even at thehigh-concentration conditions of FIG. 2 b (FIG. 2 c).

These results indicate that the acid-treated substrate can firmly adsorbDNA on the surface, and that the structure of DNA adsorbed on thesubstrate surface can be controlled by varying concentrations of DNA.

Example 3

In order to examine effects of acid treatment on DNA immobilization andextension in more detail, infrared analysis was performed using 3 kindsof solvents. FIG. 3 represents a subtractive spectrum of infraredmeasurement performed before and after acid treatment. Measurementshowed that the absorption maxima at 3500 cm⁻¹ was due to an increasedamount of hydroxy group that had been bound to the surface.

The result coincides with the previous result that showed a structuralchange from Al—O—Al to Al—O—H. To find the cause, a spectrum from thephosphate group of the DNA molecules was measured. Data were belowdetection limit (results not shown).

For the measurement of surface energy, the contact angle of Al₂O₃substrate was measured with water, diiodomethane or hexadecane, using aSessible drop method (Kyowa interface science Co., Ltd.). Ambienttemperature was maintained at 25° C. and the moisture at 40%. Averagecontact angles were found to be 5.8°, 37.5°, and 16.5° in water,diiodomethane and hexadecane, respectively. Surface free energy wascalculated according to Young-Dupre equation and extended Fowkesequation:W _(sl)=γ(1+cos θ_(sl))W _(sl)/2=(γ_(s) ^(d)·γ_(l) ^(d))^(1/2)+(γ_(s) ^(p)·γ_(l)^(p))^(1/2)+(γ_(s) ^(h)·γ_(l) ^(h))^(1/2)  [Equation 1]

Here, γs and γl represent solid surface energy and liquid surfaceenergy, respectively, and d, p, and s represent van der Waals force,dipole interaction, and hydrogen bonding, respectively.

TABLE 1 Surface energy van der Waals Dipole Hydrogen force interactionbonding Total Acid treatment 26.5 26.8 35.8 89.1 No acid 27.4 6.4 3669.8 treatment

As shown in Table 1, the surface energies due to van der Waals force,dipole interaction, and hydrogen bonding were estimated to be 26.5,26.8, and 35.8, respectively, in the chemical treated sample. The totalsurface energy was thus estimated to be about 89.0. In the untreatedsample, the surface energies were estimated to be 27.4, 6.4, and 36.0,and the total surface energy 69.8. The results indicate a quantitativeincrease of surface free energy by the action of the dipole interactioncomponent.

It was found by the measurements that the covering of the substratesurface with the hydroxy group was due to both structural changes by theacid treatment and removal of foreign substances, and that the surfaceenergy was influenced by the action of dipole interaction betweenhydroxy groups. It can be said from this that the hydrogen bondingbetween the hydroxy group on the substrate surface and the phosphategroup of the DNA plays a role in the mechanism of immobilization. Aprevious study has shown that octadecyl phosphate strongly reacts withthe Al₂O₃ surface to form a bulk (aluminoalkyl) phosphonate. Al—O—Pbonds are also formed in the same manner.

Example 4

A DNA solution was observed with a fluorescent microscope by adding afluorescent dye. To confirm effects of acid treatment, an APS-coatedglass substrate (Matsunami glass corp.) was used as a control. APStreatment has been widely used for immobilization of DNA molecules on asubstrate surface. The APS-coated glass is covered with the positivelycharged amino groups, and therefore the DNA with the negatively chargedphosphate groups around the double helix is adsorbed on the substratesurface by the electrostatic force.

Two hundred micro liters of λ DNA solution (80 ng/μl) was mixed with 1μM YO-PRO1 (Y-3603; Molecular probes Inc.). The mixture was then appliedto the acid-treated substrate and the APS-coated glass. After oneminute, the solution on the surface was dried by blowing a dry nitrogengas, and a fluorescent image was observed with an optical microscope(Olympus) (FIG. 4). FIG. 4 a shows the result of observation ofAPS-coated glass. FIG. 4 b shows the result of observation of Al₂O₃substrate.

These results showed that the immobilization of DNA was possible withoutany special modification to the substrate, and that the DNA could beimmobilized in much the same way as with the modified substrate.

The fluorescent images of DNA molecules from the APS-coated glass andthe acid-treated Al₂O₃ substrate show that the DNA has been immobilizedon the substrate surface and extended thereon. It is notable that theamount of DNA adsorbed on the Al₂O₃ substrate without any chemicalmodification was about the same as that on the APS-coated glass.

In sum, by a very simple treatment with an acid solution, the inventorsof the present invention immobilized and extended DNA molecules on theatomically flat Al₂O₃ substrate. The surface treatment with acidsolution enhanced hydrophilicity and thereby immobilization of DNA onthe surface by hydrogen bonding. Minimum structural changes are neededowning to the atomically flat surface, and the Al₂O₃ substrate isoptically transparent in the visible light range and is electricallyinsulating. This makes the simple method useful as a basic samplepreparation technique for the observation of DNA or a DNA-proteincomplex, and for the measurement of electrical mobility.

Example 5

A DNA solution and a gold nanoparticle solution were applied to Al₂O₃substrates, and adsorption on the substrates was observed (FIG. 5). FIG.5 a shows the substrate after a Poly(dA)-Poly(dT) DNA solution that hadbeen applied to the substrate was removed with a spin coater. The DNAconcentration was 250 μg/ml, and the speed of the spin coater was 800rpm. As can be seen from above, a random network structure of DNA wasformed on the Al₂O₃ substrate. FIG. 5 b shows the substrate after thegold nanoparticle solution that had been applied on the substrate wasremoved with a spin coater. The concentration of gold particles was 10¹⁵particles/ml, and the speed of the spin coater was 500 rpm. It can beseen from above that the gold particles were randomly distributed overthe Al₂O₃ substrate.

These results indicate that the adsorption of DNA and gold nanoparticleson the substrate occurs independently of the step structure of the Al₂O₃substrate.

Example 6

By spreading a mixture of DNA solution and gold fine particle solutionover the surface of the Al₂O₃ substrate, a one-dimensional arrangementof gold particles can be obtained. Samples were prepared as follows. A4:1 mixture of a DNA solution (250 μg/ml) (Poly(dA)-Poly(dT)) and asolution containing about 6×10¹³ gold particles/ml (diameter of 5 nm)was prepared. The mixture was dropped on the substrate surface and thesolution on the substrate was removed using a spin coater. FIG. 6 showsa result of observation of the substrate surface with an atomic forcemicroscope (Seiko Instruments Inc.).

As shown in FIGS. 6( a) through 6(d), the gold fine particles wereselectively arranged only on DNA portions on the step structure of theAl₂O₃ substrate. This suggests that the arrangement of gold fineparticles on the substrate surface can be controlled very easily onlywith the use of a spin coater. A preferable ratio of DNA and goldnanoparticles in the mixture was found to be 100:1 to 0.5:1 (moles ofDNA/numbers of gold nanoparticles).

FIG. 7 a schematically illustrates the result shown in FIG. 6. FIG. 7 bis a result of observation with AFM. The nanoparticles are selectivelyadsorbed on edges of the step structure of the Al₂O₃ substrate and onDNA network portions. This phenomenon is considered to be due tocapillary action. In the drying step, the water traps the nanoparticles.FIG. 8 schematically illustrates how arrangements of nanoparticles occurby capillary action.

Example 7

FIG. 9 represents procedures of a patterning process suitable for amethod for arranging fine particles on a substrate in a desired shapeaccording to the present invention. As the substrate, a glass substrateof Matsunami glass corp., prepared by treating a glass slide substratewith a poly-L-lysine coating, was used (for example, product numberSD10011, product name Poly-Lysine coat-type).

Next, using a nano-imprint device (product of OBDUCAT AB), a mold waspressed against the poly-L-lysine coating (simply “PLL coating”hereinafter) for 5 minutes under 6 MPa at a temperature of 100° C.(imprinting). At a maintained pressure (6 Mpa), the temperature waslowered to room temperature to harden the PLL coating. After hardeningthe PLL coating, the mold was separated from the substrate to obtainirregular patterns for DNA on the PLL coating.

As the mold, a Si wafer was used that had been prepared by attaching aSiO₂ thermally-oxidized film on Si. Using a stepper, the SiO₂thermally-oxidized film was patterned by lithography.

Next, about a 100 μl of an aqueous solution of powdery salmon sperm DNA(Nippon Kagaku Shiryo Kabushiki Kaisha) (1 μg/ml) adjusted with 0.3mol/l sodium chloride+0.03 mol/l sodium citrate was dropped over thesurface of the imprinted PLL-coated glass. The substrate was then heated(baked) with a hot plate for 1 hour at 80° C. to evaporate moisture andpromote immobilization of DNA and the PLL coating. This was followed byirradiation of UV light (254 nm) with a UV irradiator for 5 minutes tofurther promote immobilization of DNA and the PLL coating. Then, thesubstrate was washed first by water and then hot water (about 80° C.) toremove residual DNA remaining on the substrate surface. As a result, aself-organizing material patterning substrate was obtained.

FIGS. 10 a through 10 d are results of fluorescent microscopeobservation (Olympus, ×100) of DNA patterns immobilized on thesubstrate. The observation was made by staining DNA with a fluorescentdye dropped on the substrate. In FIGS. 10 a through 10 d, white linesindicate DNA immobilized on the substrate. FIG. 10 a shows DNAimmobilized in parallel lines. In FIG. 10 b, DNA is immobilized on sidesof square grids. FIG. 10 c shows DNA immobilized on sides of rectangulargrids. In FIG. 10 d, DNA is immobilized on sides of square grids, and ineach grid by forming rectangles.

FIGS. 11 a through 11 d show molds that have been printed withDNA-immobilizing patterns using silicon dioxide, and results ofobservation of substrate patterns after DNA has been immobilized thereonfollowing mold imprinting. FIG. 11 a shows a mold with square grids.FIG. 11 b shows a substrate that has been imprinted with the mold ofFIG. 11 a and on which DNA is immobilized. FIG. 11 c shows a mold withrectangular molds formed in square grids. FIG. 11 d shows a substratethat has been imprinted with the mold of FIG. 11 c and on which DNA isimmobilized.

Using the DNA patterns of the patterning substrates, DNA was modifiedwith gold colloids to arrange the gold colloids on the surface of DNA.First, a commercially available gold colloid solution (Tanaka KikinzokuKabushiki Kaisha, particle diameter 40 nm, concentration 0.006 wt %.)was centrifuged (15000 rpm, 1 hour). The precipitate was removed and thesolution was centrifuged again (15000 rpm, 1 hour). The resultingconcentrated gold colloids were diluted about 10 times with water toprepare a gold colloid solution. Next, the substrate with the DNApattern was dipped in the gold colloid solution for about 2 hours tomodify the DNA with the gold colloids. This was followed by taking outthe substrate from the gold colloid solution, and removing excessmoisture from the substrate surface by blowing.

FIG. 12 a shows a result of observation before gold colloidmodification. FIG. 12 b shows a result of observation of the substrateafter gold colloid modification. For observation, an atomic forcemicroscope (Seiko Instruments Inc.) was used. As shown in FIG. 12 b, thegold colloids were arranged according to the pattern of DNA immobilizedon the substrate shown in FIG. 12 a. Note that, the numbers “0.00-153.92nm” and “0.00-276.74 nm” respectively shown at the bottom of FIGS. 12 aand 12 b indicate heights, corresponding to the shade of horizontal barsshown above these numbers.

Comparative Example

Ten micro liters of Poly(dA)-Poly(dT) solution (1250 ng/μl) was mixedwith 15 μl of Au fine particle solution to prepare a mixed solution ofPoly(dA)-Poly(dT) (500 ng/μl) and 5-nm gold fine particles (1×10¹⁴particles/ml). The mixture was allowed to stand overnight at 4° C. Thesolution was dropped on acid-treated Al₂O₃ substrate, which was thenspun with a spin coater at 500 rpm for 1 minute to remove excesssolution. FIG. 13 a shows a result of observation of a substratesurface. Under these conditions, there was no selective adsorption ofthe gold particles on the step edges, but the gold fine particles wererandomly adsorbed on the substrate.

Next, by mixing a λ DNA solution (300 ng/μl) and a Au fine particlesolution at a 1:1 ratio, a mixed solution was prepared containing λ DNA(150 ng/μl) and 5-nm gold fine particles (×10¹⁴ particles/ml). Themixture was allowed to stand overnight at 4° C. The solution was droppedon acid-treated Al₂O₃ substrate, which was then spun with a spin coaterat 500 rpm for 1 minute to remove excess solution. FIG. 13 b shows aresult of observation of a substrate surface. Under these conditions,there was no selective adsorption of the gold particles on the stepedges, but the gold fine particles were randomly adsorbed on thesubstrate.

The foregoing showed that the fine particles could be orderly arrangedon the substrate, preferably at a DNA concentration of 250 ng/μl and arotational speed of spin coater at 500 rpm.

The embodiments and concrete examples of implementation discussed in theforegoing detailed explanation serve solely to illustrate the technicaldetails of the present invention, which should not be narrowlyinterpreted within the limits of such embodiments and concrete examples,but rather may be applied in many variations within the spirit of thepresent invention, provided such variations do not exceed the scope ofthe patent claims set forth below.

INDUSTRIAL APPLICABILITY

The present invention enables a hydroxy group to be introduced onto asurface of an acid-treated metal oxide (for example Al₂O₃, ZnO, TiO₂)substrate, and thereby allows DNA to be directly and firmly bonded tothe substrate via the hydroxy group. The present invention can be usedto form individual molecules of extended DNA or a network structure ofDNA bundles on a substrate surface. Conventionally, it has beendifficult to control arrangements of gold fine particles over a widearea. With the present invention, arrangement control of gold fineparticles is possible over a wide area of the substrate.

The present invention is highly useful in applications such as structureanalysis and DNA electronics. The invention is applicable toconstruction of nanoscale circuits, functional conductive materials, andphotomasks.

1. A method for arranging fine particles on a substrate of metal-oxide,comprising the steps of: applying an acid solution to the substrate;obtaining a mixed solution by mixing a solution containing the fineparticles with a solution containing a self-organizing material;applying the mixed solution to the substrate after removing the acidsolution from the substrate; and drying the mixed solution applied tothe substrate with the self-organizing material still present on thesubstrate.
 2. The method as set forth in claim 1, wherein theself-organizing material is a nucleic acid, a protein, an amino acid, alipid, or a sugar.
 3. The method as set forth in claim 1, wherein themetal oxide is Al₂O₃, ZnO, TiO₂, SiO₂, ZrO₂, SrTiO₂, LaAlO₃, Y₂O₃, MgO,GGG, YIG, LiTaO, LiNbO, KTaO₃, KNbO₃, or NdGaO₃.
 4. The method as setforth in claim 1, wherein the fine particles comprise gold, silver,platinum, palladium, iridium, rhodium, osmium, ruthenium, nickel,cobalt, indium, copper, TiO₂, or BaTiO₃.
 5. The method as set forth inclaim 1, wherein the fine particles have a particle diameter in a rangeof 1 nm to 100 nm.
 6. The method as set forth in claim 1, wherein thedrying step is performed by blowing a dry inert gas or air.